Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS), manufactured for a variety of industrial and consumer applications, are ubiquitous environmental pollutants. Their accumulation in humans and wildlife raises serious health concerns. Here, we examined the potential effects of PFOA and PFOS on the innate immune system in mice. Short-term dietary exposure to high doses reduces the total number and subpopulations of circulating white blood cells. Moreover, production of proinflammatory cytokines by macrophages in the peritoneal cavity and bone marrow, but not in the spleen following exposure to in vitro or in vivo stimulation by bacterial lipopolysaccharides is enhanced. With respect to adaptive immunity, PFOS reduces the total numbers of thymocytes and splenocytes and subpopulations thereof in a dose dependent fashion. Furthermore, comparison of wild-type mice and the corresponding knock-out strain lacking peroxisome proliferator-activated receptor-alpha revealed that these immunological changes are partially dependent on this receptor. Our further studies also show that sub-chronic dietary exposure to an environmentally relevant dose of PFOS does not alter the cellularity of the thymus and spleen and exerts no influence on humoral immune responses. To facilitate examination of the effects of PFOA and PFOS on the hepatic immune system, we developed a procedure for mechanical disruption that yields a larger number of functionally competent immune cells from this organ. In our last study, lower doses of PFOA or PFOS induced hypertrophy of hepatocytes and altered the hepatic immune status. Thus, we find that short-term, high- and low-dose exposure of mice to these fluorochemicals is immunohepatotoxic. / Perfluorooktanat (PFOA) och perfluorooktansulfonat (PFOS) som tillverkas för många olika industri och konsumentprodukter, är globalt förekommande miljögifter. Deras ackumulering i människor och djur ger upphov till en stark oro för hälsoproblem. Vi har granskat effekterna av PFOA och PFOS på det medfödda, ospecifika immunförsvaret. Exponering för höga doser via maten under kort tid minskar det totala antalet cirkulerande vita blodkroppar samt delpopulationerna.. Immunsvaret ökar dock efter stimulering med bakteriella lipopolysaccharider både in vitro och in vivo , dvs produktionen av proinflammatoriska cytokiner av makrofager i bukhålan och benmärgen, men inte i mjälten ökar.. När det gäller adaptiv, specifik immunitet minskar PFOS det totala antalet tymocyter och splenocyter och deras olika subpopulationer. Vid exponering för lägre doser av PFOS induceras hepatomegali utan att påverka tymus eller mjälten.   Vi kunde visa att peroxisomal proliferator-aktiverad receptor-alfa medierar effekterna utav PFOS i tymus samt delar av effekterna av PFOS i mjälten genom att använda möss som saknade denna receptor. . Dettastöds av vår studie med subkronisk exponering för en miljömässig dos av PFOS vilken inte ändrade den cellulära sammansättningen i vare sig  tymus eller mjälte och inte hade  något inflytande på det humorala immunsvaret. För att underlätta studier av hur PFOA och PFOS påverkar immunsystemet i levern utvecklade vi en metod för framrening av immunceller via mekanisk sönderdelning av levern, vilket gavett större antal av funktionella  immunceller från detta organ. I vår sista studie kunde vi påvisa att lägre doser av PFOA eller PFOS inducerade hypertrofi av hepatocyter samt en påverkan av leverns immunförsvar.

学位授与大学：京都大学 ; 取得学位: 博士(工学) ; 学位授与年月日: 2007-09-25 ; 学位の種類: 新制・課程博士 ; 学位記番号: 工博第2850号 ; 請求記号: 新制/工/1419 ; 整理番号: 25535 / Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13379号 / 工博第2850号 / 新制||工||1419(附属図書館) / 25535 / UT51-2007-Q780 / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 藤井 滋穂, 教授 伊藤 禎彦 / 学位規則第4条第1項該当

This thesis is focussed on the investigation of matrices for matrix-assisted diffusion-ordered spectroscopy (MAD). Diffusion-ordered spectroscopy (DOSY) is a family of experiments where the resonances in the chemical shift dimension are further dispersed in an extra dimension according to diffusion coefficient. A typical DOSY spectrum shows one single diffusion coefficient for all the resonances coming from one single species. However, If two or more resonances overlap, the diffusion resolution of the DOSY spectrum is compromised and a spurious diffusion coefficient results, intermediate between the species. In case of signal overlap, the use of more advanced processing methods aids to separate two analytes that differ by at least 30% in diffusion coefficient. In practice, many mixtures contain species of similar diffusion coefficients whose resonances overlap in the chemical shift dimension. The addition of co-solutes can modify the chemical environment (matrix), with which different analytes interact to different extents, and enhance the diffusion resolution of DOSY. However, the addition of co-solutes can risk the benefits of DOSY by increasing the probability of signal overlap. Signal overlap in MAD is avoided by using a 1H NMR-invisible surfactant such as sodium perfluorooctanoate (NaPFO), which has replaced each proton by a fluorine atom. PFO micelles are a tunable matrix which allows the separation of analytes via coulombic interactions by adjusting the pH. Differences in diffusion coefficient in NaPFO solution can be analysed using a modified Lindman's law to model the diffusion coefficient as a function of pH. The model rationalises the binding constants of analytes to PFO micelles with good accuracy, subject to the spectral data quality. Another alternative to resolve diffusion coefficients using the invisible MAD approach is by means of a commercially available alkyl surfactant like cetyltrimethylammonium bromide (CTAB). CTAB in high ionic strength solution forms worm-like micelles whose resonances can be filtered out from the final DOSY spectrum. CTAB worm-like micelles have short transverse relaxation times compared to all of the analytes in the mixture. If a transverse relaxation filter is positioned at the beginning of a standard DOSY pulse sequence, as in PROJECT-Oneshot, the strong CTAB signals vanish and leave behind only the analyte resonances and hence avoid signal overlap. Finally, the use of bovine serum albumin (BSA) as a potential invisible matrix, using a similar approach to CTAB worm-like micelles is investigated, using a relaxation-weighted DOSY pulse sequence to suppress most of the BSA background signal (at a cost in analyte signal to noise ratio). An alternative to suppress most of the BSA background and preserve most of the analyte signal is by means of mild transverse relaxation filtration and spectral editing to obtain an edited DOSY spectrum that shows only the analyte signals. Nonetheless, it is a shame that useful MAD results can only be obtained under a narrow set of conditions: i) different mole ratios BSA: analyte to aid diffusion resolution, ii) mild T2 filtration to improve analyte signal to noise ratio and iii) spectral editing to remove residual BSA background.

Les substances perfluoroalkylées (PFAS) sont des composés synthétiques utilisés dans une multitude de domaines pour leurs propriétés hydrofuges, antiadhésives et antitaches exceptionnelles. Cependant, ces contaminants, dont la neurotoxicité a été démontrée dans les études in vitro et in vivo, sont capables de traverser la barrière placentaire et d’atteindre le fœtus en développement. Bien qu’une multitude d’études épidémiologiques aient été conduites pour examiner l’association entre l’exposition prénatale aux PFAS et le neurodéveloppement des enfants, il n’y a pas de consensus dans la littérature : certaines rapportent des associations délétères, et d’autres protectrices ou nulles. Le but de cette étude est d’évaluer l’association entre l’exposition aux PFAS chez les femmes enceintes et le développement neurocomportemental et social des enfants pendant la petite enfance. Nous avons également examiné si le lien différait entre les filles et les garçons. Les données de l’étude Maternal-Infant Research on Environmental Chemicals (MIREC), une cohorte de grossesse pancanadienne, ont été utilisées. L’exposition prénatale à trois PFAS (acides perfluorooctanoïque (PFOA), perfluorooctanesulfonique (PFOS) et perfluorohexane sulfonique (PFHxS)) ainsi que leur somme (ΣPFAS) a été mesurée dans le plasma maternel prélevé durant le premier trimestre de grossesse. Lorsque les enfants étaient âgés de trois-quatre ans, les mères ont été invitées à remplir deux questionnaires sur leurs enfants : le Behaviour Assessment System for Children–2 (BASC-2), pour évaluer les difficultés émotionnelles et comportementales, et le Social Responsiveness Scale–2 (SRS-2), afin d’évaluer le développement social. À partir des données de 794 paires mère-enfant, des analyses de régressions linéaires multiples, avec ajustement pour des facteurs de confusion, ont été réalisées, et les coefficients d’association ont été calculés pour un doublement des concentrations de PFAS. La modification des associations selon le genre a été examinée au moyen de termes d'interaction et d'analyses stratifiées. Bien que la majorité des associations obtenues soient nulles, pour l’ensemble du groupe étudié, un doublement de l’exposition prénatale aux PFOS était lié à moins de déficits de motivation sociale (β = -1.03; IC : -1.88, -0.17) et le PFHxS à plus de comportements atypiques (β = 0.57; 0.04, 1.11). Cependant, les analyses selon le genre ont révélé que chez les garçons seulement, un doublement de l’exposition prénatale aux PFOA était significativement associée à des scores plus faibles pour les sous-échelles suivantes : indice des symptômes comportementaux, problèmes d'externalisation, agressivité et hyperactivité (β allant de -1.87 à -1.32). Le PFOS et la ΣPFAS étaient aussi liés à moins d’agressivité chez les garçons (β = 1.20; -2.27, -0.13 et β = -1.35; -2.55, -0.15 respectivement). À l’inverse, chez les filles, le PFOA était significativement lié à plus de symptômes d’anxiété, et le PFHxS et la ΣPFAS étaient liés à plus de problèmes de cognition sociale (β allant de 0.90 à 1.81). Dans l’ensemble, les données suggèrent que l’association entre l’exposition prénatale aux PFAS et le développement neurocomportemental et social des enfants semble différer selon le genre : un effet protecteur est observé chez les garçons, tandis qu’il ressort délétère chez les filles. Les résultats obtenus dans cette cohorte canadienne de grande taille corroborent ceux notés dans certaines études épidémiologiques rapportant un lien délétère, lequel est surtout observable chez les filles. / Perfluoroalkyl substances (PFAS) are synthetic compounds used in a wide range of fields for their exceptional water-repellent, non-stick, and stain-resistant properties. However, these contaminants, whose neurotoxicity has been demonstrated in in vitro and in vivo studies, can crossthe placental barrier, and reach the developing fetus. Although numerous epidemiological studies have been conducted to examine the association between prenatal exposure to PFAS and the neurodevelopment in children, there is no consensus in the literature: some report deleterious associations, while others report protective or null associations. The aim of this study is to investigate the association between PFAS exposure in pregnant women and the neurobehavioral and social development of children during early childhood. We also examined whether the association differed between girls and boys. We used data from the Maternal-Infant Research on Environmental Chemicals (MIREC) study, a pan-Canadian pregnancy cohort. Prenatal concentrations of three PFAS (perfluorooctanoic (PFOA), perfluorooctanesulfonic (PFOS) and perfluorohexanesulfonic acids (PFHxS)), as well as their sum (åPFAS), were measured in maternal plasma collected during the first trimester of pregnancy. When the children were three to four years old, mothers were asked to complete two questionnaires about their children: the Behaviour Assessment System for Children-2 (BASC-2) to assess emotional and behavioural difficulties, and the Social Responsiveness Scale-2 (SRS-2) to assess social development. Using data from 794 mother-child dyads, multiple linear regression analyses, with adjustment for confounding factors, were performed and regression coefficients were estimated to assess whether there was an association between each doubling of PFAS concentrations and test scores. Effect modification by child gender was examined using interaction terms and stratified analyses. For the entire study group, although most of the associations found were null, a doubling of prenatal PFOS exposure was linked to fewer social motivation deficits (β = -1.03; CI: -1.88, -0.17), and increased PFHxS was linked to more atypical behaviors (β = 0.57; 0.04, 1.11). However, gender-stratified analyses revealed that in boys only, each doubling of prenatal PFOA exposure was significantly associated with lower scores on the following BASC-2 subscales: Behavioral Symptoms Index, Externalizing Problems, Aggressivity and Hyperactivity (β ranging from -1.87 to -1.32). PFOS and åPFAS were also associated with less aggression in boys (β = 1.20; -2.27, -0.13 and β = -1.35; -2.55, -0.15 respectively). Conversely, in girls only, PFOAwas significantly associated with more symptoms of anxiety, and PFHxS and ∑PFAS were associated with more social cognition problems (β ranging from 0.90 to 1.81). Overall, the data suggest that the association between prenatal PFAS exposure and the neurobehavioral and social development of children appears to differ by gender: a protective effect is observed in boys, while a detrimental effect is seen in girls. The results obtained in this large Canadian cohort are consistent with findings from some epidemiological studies reporting a harmful link predominantly in girls.